1. Field of the Invention
The invention relates to a method for efficient and extremely simple and cost-advantageous parallel isolation of double-stranded and single-stranded nucleic acids.
2. Discussion of the Background
The parallel isolation of genomic DNA and cellular total RNA from a biological sample is connected with only a few practicable methods at the present time. A method for the separation of genomic DNA and cellular total RNA by way of selective precipitation steps, using lithium chloride, is described by Raha, S., Merante, F., Proteau, G. and Reed, J. K. (GATA, 1990, 7(7): 173-177). Another possibility for parallel isolation of DNA and RNA utilizes ultra filtration techniques, using a cesium chloride gradient for pelleting the RNA, in combination with subsequent dialysis of the DNA (Coombs, L. M., Pigott, D., Proctor, A., Eydmann, M., Denner, J. and Knowles, M. A.; Anal. Biochem. (1990); 188; 338-343). The published method is extremely time-consuming and requires a special effort in terms of apparatus.
A disadvantage of this method, however, is the complicated preparation steps and the use of highly toxic and carcinogenic substance groups such as chloroform and phenol. Finally, the time required is also very great.
A simplified method for simultaneous isolation of DNA and RNA is disclosed in the Laid Open patent application WO 97/28171 A1. Here, the biological sample is lysed using a chaotropic buffer. After lysis, the addition of nanoparticles (smaller than 40 nm) made of monodisperse silicon material takes place. The genomic DNA binds to these particles. The batch is then centrifuged. This causes the silicon particles to be pelletized. The excess is transferred to a new reaction vessel. The pelletized carrier material is subsequently washed with a washing buffer that contains ethanol, and the bound DNA is finally dissolved from the mineral carrier material again, using a low-salt buffer. The remaining excess is subjected to phenol/chloroform extraction, in traditional manner, and the RNA is finally precipitated in this way, and dissolved using water, after washing steps.
The method is faster than the method described above. However, in this method, as well, work is carried out with highly toxic and carcinogenic substance groups such as chloroform and phenol.
Another method for separation and isolation of single-stranded and double-stranded nucleic acids is disclosed in the patent EP 1 146 049 B1.
The method is based on the treatment of a source that contains nucleic acids, using at least one mineral carrier material, in such a form that
1. the single-stranded nucleic acid is isolated, in that the treatment conditions are adjusted by means of an aqueous mixture of chaotropic salts and low aliphatic alcohols, in such a manner that subsequently, the single-stranded nucleic acid is primarily adsorbed onto a mineral carrier, while the double-stranded nucleic acid is not adsorbed. The bound, single-stranded nucleic acid is subsequently washed and finally eluted from the carrier material by means of a low-salt buffer.
2. the double-stranded nucleic acid is isolated, in that the treatment conditions are adjusted by means of a mixture of substances that complex earth-alkali ions, without low aliphatic alcohols, in such a manner that subsequently, the double-stranded nucleic acid is primarily adsorbed onto a mineral carrier, while the single-stranded nucleic acid is not adsorbed. The bound, double-stranded nucleic acid is subsequently washed and finally eluted from the carrier material by means of a low-salt buffer.
3. the double-stranded nucleic acid is isolated in that the treatment conditions are adjusted by means of the presence of a sarcosinate but without low aliphatic alcohols, in such a manner that subsequently, the double-stranded nucleic acid is primarily adsorbed onto a mineral carrier, while the single-stranded nucleic acid is not adsorbed. The bound, double-stranded nucleic acid is subsequently washed and finally eluted from the carrier material by means of a low-salt buffer.
4. the double-stranded or single-stranded nucleic acid is isolated in that the treatment conditions are adjusted by means of an aqueous mixture of chaotropic salts and low aliphatic alcohols, in such a manner that both nucleic acid fractions adsorb onto a mineral carrier. Separation of the nucleic acids takes place by means of selective elution. In this connection, the double-stranded nucleic acid is dissolved from the carrier material by means of a solution having a reduced ion strength and low aliphatic alcohols. The remaining single-stranded nucleic acid is subsequently washed and finally dissolved from the carrier material by means of a low-salt buffer.
Alternatively to this, the single-stranded nucleic acid can be selectively eluted from the carrier material by means of a solution that contains substances that complex earth-alkali ions and/or contains sarcosinates. The now remaining double-stranded nucleic acid is subsequently washed, once again, and finally dissolved from the carrier material by means of a low-salt buffer.
The method is simple in its implementation, and does entirely without the use of phenol or chloroform. Also, time-consuming ethanol precipitation is not required.
However, it has also been shown that often, there is no selectivity of isolation of the nucleic acids, in each instance. For example, a commercially available extraction kit for isolation of RNA on the basis of the method described always requires DNase digestion. This is required since the selectivity for isolation of RNA solely with the method described, as presented, is not sufficient. The double-stranded DNA must additionally be removed by means of an enzymatic method.
WO 97/37040 A2 also refers to a method for separating single-stranded and double-stranded nucleic acids, particularly with regard to the isolation of HCV RNA from bodily fluids. As was already disclosed in EP 1 146 049 B1, binding of the double-stranded nucleic acid to a silica material also takes place in WO 97/37040 A2 in that the buffer used for this purpose requires a high concentration of EDTA (at least 100 mM). Thus, the presence of a complex-forming agent is required, at a high concentration, in order to bind double-stranded nucleic acid to mineral silica particles, and to prevent binding of single-stranded nucleic acid.
Thus, there has been a continued need for a method for efficient and extremely simple and cost-advantageous parallel isolation of double-stranded and single-stranded nucleic acids that avoids the above disadvantages.